3-aminoethyl-n-amidino-2,5-dihydropyrrole derivatives having arginine mimetic properties

ABSTRACT

The present invention ocncerns new amidinopyrroline derivatives of formula (I) with the meanings of the symbols as given in the description as well as a process for their production and their use in pharmaceutical preparations.

[0001] The present invention concerns new amidinopyrroline derivatives,processes for their production and their use thereof in pharmaceuticalpreparations.

[0002] It was found that compounds of the general formula (I) have theproperties of an arginine mimetic. Arginine mimetics are pharmacophoreswhich can replace arginine or an arginyl residue for example ininhibitors. Derivatives of (I) can replace arginine or already knownarginine mimetics in biologically active substances and especially intherapeutically active substances. In particular they can be used ininhibitors for serine proteases such as thrombin or trypsin inhibitors.Inhibitors of thrombin inhibit the thrombin induced coagulation offibrinogen in blood as well as the thrombin induced aggregation of bloodplatelets. Thus they prevent the formation of coagulation thrombi andplatelet-rich thrombi and can be used to treat and prevent diseases suchas thromboses, apoplexy, cardiac infarction, inflammations andarteriosclerosis

[0003] Thrombin the last enzyme of the coagulation cascade cleavesfibrinogen to fibrin which is cross-linked by factor XIII and becomes aninsoluble gel which forms the matrix for a thrombus. Thrombin activatesplatelet aggregation by proteolysis of its receptor on the bloodplatelets and in this manner also contributes to thrombus formation.When blood vessels are damaged these processes are necessary to stopbleeding. Under normal circumstances no measurable thrombinconcentrations in blood plasma are present. An increase of the thrombinconcentration can lead to the formation of thrombi and thus tothromboembolic diseases which occur very frequently above all inindustrial countries. Thrombin is kept ready in the plasma in the formof prothrombin and is released from this by factor Xa. Thrombinactivates factors V, VII and XI which then convert factor X into Xa. Bythis means thrombin catalyses its own release which is why very rapidincreases in thrombin concentrations can occur. Thrombin inhibitors andfactor Xa inhibitors can therefore inhibit the release of thrombin, andthe platelet induced and plasmatic blood coagulation

[0004] Trypsin is a digestive enzyme which is excreted by the pancreaswhen required. When the pancreas is damaged or inflamed the trypsinrelease which this causes can lead to tissue destruction. Trypsininhibitors can reduce this threat and be used for instance to treatpancreatitis.

[0005] In addition such arginine mimetics can be incorporated intoactive substances which are able to inhibit the binding of ligands thatbind to their receptor via sequences containing RGD. RGD stands for thetripeptide Arg-Gly-Asp. Such ligands are for example fibrinogen,vitronectin or fibronectin. Such active substances can be used to treatdiseases which are due to thrombo-embolic events such as stroke,myocardial infarction or arterial occlusive diseases as well asinflammations, osteoporosis or tumour diseases.

[0006] The invention concerns compounds which contain the structuralelement I as a pharmacophore as well as modifications known to a personskilled in the art which can be derived from the parent substance ofstructure I,

[0007] in which R1, R2 and Y can be the same or different and denotehydrogen or an organic residue. Preferred organic residues are suchwhich result in a biological active compound of general formula I.Biological active means inter alia substances for plant protection andpreferred pharmacological active compounds. Prodrugs of such biologicalactive compounds are also included in the preferred structures offormula I. Prodrugs are substances which will be metabolized in vivointo the biological active compound. An example, but not a limitationfor prodrugs are esters which will be converted to free acids by theorganism, e.g. by the liver metabolism

[0008] Especially preferred are such derivates of compounds of thegeneral formula I, wherein an arginine substructure or a known argininemimetic substructure in a biological active sustance is substituted bythe backbone structure of formula I′ or I″.

[0009] wherein the jagged bonds represent in said biological activecompound the positions of the optional chemical bonds of the arginine orknown arginine mimetic substructure which is substituted by the argininemimetic structure (I′) or (I″) of the invention

[0010] A collection of pharmacological active compounds can be found indata bases for INNs (International Nonproprietary Names) which arehereby incorporated by reference.

[0011] In particular compounds of the general formula (I) are preferred

[0012] in which R1, R2 denote hydrogen, an amino acid, peptidyl,alkylsulfonyl or arylsulfonyl residue,

[0013] Y denotes hydrogen or a residue of the formula COX,

[0014] X denotes hydrogen or an OR3 or NR1′R2′ residue,

[0015] R3 denotes hydrogen or lower alkyl such as methyl, ethyl, propylor butyl preferably ethyl and

[0016] R1′, R2′ can be the same or different and have the meanings ofthe residues R1 and R2.

[0017] Amino acid residue usually denotes the residue of a natural orunnatural amino acid. Unnatural amino acids are understood as α-, β-, γ-and ω-aminocarboxylic acids as well as derivatives thereof Derivativesare in particular understood as compounds which are alkylated on theamino group or the carboxy group. Derivatives are also included whichare either decarboxylated or deaminated.

[0018] Examples of such amino acids and derivatives thereof are statedin the examples and preferred compounds. In particular these are D-aminoacids, citrulline, homocysteine, homoserine, hydroxyproline,hydroxylysine, ornithine, sarcosine, tranexamic acid, Adc[3-(2-aminoethyl)-2,5 dihydropyrrol-1-yl]-carbamidine], Ada[(1-amidino-2,5-dihydro-1H-pyrrol-3-yl)-alanine], Cha[cyclohexyl-alanine], Choi [2-carboxy-6-hydroxy-octahydroindol],norLeu(cyclo)-Gly [3-amino-2-oxo-hexahydro-1-azepine-acetic acid], Pcs[4-piperidine carboxylic acid], Pip [pipecolic acid], Pla [phenyllacticacid], N-Me-Phe, HOOCCH2-Phe, HOOCCH2-Cha,1-carboxy-perhydroiso-quinoline, N-cyclopentylglycine, EtSO2-Phe,N-(BuSO2-NorLeu(cyclo)-Gly.

[0019] A peptidyl residue is understood as a residue composed of anydesired number of natural or unnatural identical of different aminoacids. Peptidyl residues with 1-50 amino acids are preferred and thosewith 1, 2, 3 or 4 amino acids are particularly preferred.

[0020] Alkyl usually denotes a linear or branched alkyl residue with oneto six carbon atoms. Aryl usually denotes a carbocycle with 6 to 14 Catoms or a 5- or 6-membered heterocycle with 1, 2 or 3 heteroatomsselected from O, N or S. Unsubstituted or optionally substituted phenylor naphthyl residues are preferred.

[0021] The arginine mimetic (I) in which R1 and R2 denote hydrogen isproduced by well-known methods. Production from the precursor (II) isadvantageous,

[0022] in which the primary amino group is protected preferably byreaction with phthalic acid anhydride to form the phthalimide, then thesecondary amino group is amidated preferably by the methods described inBannard et al., Can J Chem. 1958, 1541 and subsequently the phthalimidogroup is cleaved preferably by hydrazine hydrate and subsequenttreatment with hydrochloric acid.

[0023] Compounds of the formula (II) in which Y denotes hydrogen can beproduced by

[0024] a) reducing the amide group of a compound of formula (III),

[0025] preferably with lithium aluminium hydride or lithium borohydridein the presence of trimethylchlorosilane or

[0026] b) by rearranging the exocyclic double bond of a compound of thegeneral formula (IV)

[0027] in which R5 denotes a protecting group for example a benzoylgroup, an alkyloxycarbonyl group or a benzyloxy-carbonyl group into theisomer with an endocyclic double bond and subsequently cleaving theprotecting groups. The rearrangement of the exocyclic double bond to theendocyclic double bond is carried out in the presence of lyes preferablysodium hydroxide solution as described analogously in M. I. Labouta etal., Acta Chem. Scand. Ser. B. 1982, 669 - 674. This is followed by thecleavage of the protecting groups R5 and of the phthalimido group.

[0028] c) to use the process as shown in scheme 1

[0029] Compounds of the general formula (II) in which Y denotes acarboxyl group can be produced according to the process outlined inschemes 2 or 3.

[0030] The compound (III) is produced from compounds of the generalformula (XIV)

[0031] wherein R5 has the above-mentioned meanings with a reagent thatactivates the carboxyl group for example thionyl chloride orchloroformic acid isobutyl ester followed by ammonia. Subsequently theprotecting group R5 is cleaved preferably with hydrochloric acid indioxane or with trifluoroacetic acid or with hydroden bromide in glacialacetic acid. Compounds of the general formula (XIV) are known forexample from M. I. Labouta et al., Acta Chem. Scand. Ser. B, 1982, 669 -674 or can be produced by the processes described in this publication

[0032] The compounds of the general formula (IV) are produced byreacting compounds of the general formula (XV) with the compound (XVI)according to a Wittig reaction

[0033] wherein R denotes for a protecting group.

[0034] This Wittig reaction and the reagent (XVI) are described in Ch.Sellier et al., Liebigs Ann Chem 1992, 317-324. The compounds (XV) aredescribed in A. G. Schultz, Tetrahedron 1980, 1757-1761.

[0035] Comments on scheme 2.

[0036] R3 and R5 have the above-mentioned meanings;

[0037] R6 denotes an alkyl or aryl residue such as a methyl, ethyl,trifluoromethyl, phenyl, tosyl or 4-nitro-phenyl residue, preferably amethyl or tosyl residue.

[0038] L usually denotes a sulfonic acid residue such as amethanesulfonic acid, trifluoromethanesulfonic acid or p-toluenesulfinicacid residue, or a halogen such as chlorine, bromine, iodine or actate;

[0039] MHal denotes a metal halogenide such as NaCl, NaBr, Kl, MgCl2 orMgBr2,

[0040] compounds of formula (V) are described in Timothy L. et al., JOrg. Chem 48. 1129-1131 (1983),

[0041] Conversion of an alcohol of formula (V) into a sulfonic or aceticacid ester of formula (VI) is achieved by standard methods of organicchemistry.

[0042] The conversion of an alcohol of formula (V) into a halogenide offormula (VI) by means of N-chloro-succinimide, N-bromosuccinimide orN-iodosuccinimide (NCS, NBS, NIS) in the presence of triphenylphosphine(Ph3P) is carried out analogously to corresponding literature methods(e.g. Rozwadowska M. D, Tetrahedron Asym, 4, 1619-1624(1993));

[0043] A compound of formula (X) is converted into a compound of formula(II) by means of Claisen rearrangement analogously to methods which aredescribed in Kazmaier U et al., Tetrahedron 52, 941-954 (1996),

[0044] The epoxide opening of an epoxide of formula (XII) to form anallyl alcohol of formula (XI) is carried out under conditions which aredescribed in Joshi V S et al. Tetrahedron, 24, 58'7-5830 (1968),

[0045] Compounds of formula (XIII) are described in Grubbs H et al., J.Am Chem. Soc, 114, 7324-7325 (1992),

[0046] The decarboxylation of malonic esters by means of metalhalogenides in dimethylsulfoxide and at a high temperature is describedin Krapcho A P et al Tetrahedron Lett. 957 (1973).

[0047] Some of the compounds of the general formula (I) have one orseveral asymmetric centres. Hence optically active forms are also asubject matter of the invention as well as tautomers.

[0048] The invention also concerns all salts of compounds of the generalformula (I) Salts are primarily the acid addition salts. Physiologicallyacceptable salts come mainly into consideration for pharmaceuticalpurposes. Examples of salts of the compound of formula (I) which can beused physiologically are salts with physiologically tolerated mineralacids such as hydrochloric acid, sulfuric acid, sulfurous acid orphosphoric acid or with organic acids such as methane-sulfonic acid,p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid,fumaric acid, maleic acid, tartaric acid, succinic acid or salicylicacid.

[0049] For the production of pharmaceutical agents the substances of thegeneral formula (I) and their salts are admixed with suitablepharmaceutical carrier substances, aromatics, flavourings and dyes andare for example formed as tablets or dragees or are suspended ordissolved in water or oil for example in olive oil with addition ofappropriate auxiliary substances.

[0050] The substances of the general formula (I) and their salts can beadministered enterally or parenterally in a liquid or solid form. Wateris preferably used as the injection medium which contains the usualadditives in injection solutions such as stabilizing, agents,solubilizers or buffers Such additives are for example tartrate andcitrate buffer, complexing agents (such as ethylenediamine tetraaceticacid and non-toxic salts thereof) and high molecular polymers such asliquid polyethylene oxide to regulate viscosity Solid carriers are e.g.starch, lactose, mannitol, methylcellulose, talcum, highly-dispersedsilicic acids, high molecular fatty acids (such as stearic acid) animaland vegetable fats and solid high molecular polymers (such aspolyethylene glycols). Preparations which are suitable for oraladministration can optionally contain flavourings and sweeteners

[0051] The compounds are usually administered in amounts of 10-1500 mgper day with regard to 75 kg body weight. It is preferred to administer1-2 tablets with a content of active substance of 5-500 mg 2-3 times perday The tablets can also be retarded in which case only 1-2 tablets with20-700 mg active substance have to be administered once per day. Theactive substance can also be applied by injection 1-8 times per day orby continuous infusion in which case 50-2000 mg per day are usuallyadequate

DESCRIPTION OF THE FIGURES

[0052]FIG. 1 Spatial structure of Adc from D-Pla-D-Phe-L-Choi-Adc (thickrods) in the enzyme trypsin (thin rods) from the X-ray structure ofexample 2. The spatial structure of the inhibitor DFPR in human thrombinis superimposed over this (inhibitor: thin rods with spheres, thrombin:thin lines). The sphere represents a water molecule

[0053] The following abbreviations are used in the examples:

[0054] Ac=acetyl

[0055] Ada=(1-amidino-2,5-dihydro-1H-pyrrol-3-yl)-alanine

[0056] Adc=[3-(2-aminoethyl)-2,5-dihydropyrrol-1-yl]-carbamidine

[0057] Asp=aspartic acid

[0058] Bn=benzyl

[0059] Boc=tert.butyloxycarbonyl

[0060] Bu=butyl

[0061] Cbz=benzyloxycarbonyl

[0062] Cha=cyclohexylalanine

[0063] Choi=2-carboxy-6-hydroxy-octahydroindole

[0064] DBU=1,8-diazabicyclo[5 4.0]undec-7-ene

[0065] DIBAL-H=di-isobyle aluminum hydride

[0066] DMAP=4-Dimethylaminopyridine

[0067] DMF=dimethylformamide

[0068] eq=equivalent

[0069] Et=ethyl

[0070] Fmoc=9-Fluorenylmethoxycarbonyl

[0071] Gly=glycine

[0072] HMDS=hexamethyldisilazane

[0073] i-Pr=iso-propyl

[0074] Me=methyl

[0075] NMM=N-methylmorpholine

[0076] norLeu(cyclo)-Gly=3-amino-2-oxo-hexahydro-1-azepine-acetic acid

[0077] Pcs=4-piperidine carboxylic acid

[0078] Ph=phenyl

[0079] Phe=phenylalanine

[0080] Pip=pipecolic acid

[0081] Pla=phenyllactic acid

[0082] Pmc=2,2,5,7,8-pentamethylchroman-6-sulfonyl

[0083] Pro=proline

[0084] py=pyridine

[0085] Ser=serine

[0086] t-Bu=tert.-Butyl

[0087] TBTU=2-(1H-benzotriazol-1-yl)-1.1 3.3-tetramthyluroniumtetrafluorborate

[0088] TCP=Tritylchloride-polystyrene

[0089] THF=tetrahydrofuran

[0090] Troc=2,2,2-Trichlorine-ethoxycarbonyl

[0091] Tyr=tyrosine

[0092] Trp=tryptophane

[0093] t_(R)=retention time

[0094] Val=valine

[0095] The C,N,O-backbone of Ada and Adc are the backbone structureswhich substitute the arginine or arginine mimetica structure inbiological active substances of the invention, wherein OH-function ofthe carboxylic acid group of Ada may be replaced by another backboneatom, e.g. C, N or S.

[0096] Apart from the compounds mentioned in the examples and compoundsderived by combining all meanings of the substituents stated in theclaims the following are preferred within the sense of the presentinvention:

[0097] 1. N-Me-D-Phe-Cha-Adc

[0098] 2. HOOCCH2-D-Phe-Pro-Adc

[0099] 3. HOOCCH2-D-Cha-Pip-Adc

[0100] 4. 1-Carboxy-perhydroisoquinolinyl-Pro-Adc

[0101] 5. 1-Carboxy-perhydroisoquinolinyl-N-cyclopentyl-Gly-Adc

[0102] 6 EtSO2-D-Phe-Pro-Adc

[0103] 7 EtSO2-D-Phe-N-cyclopentyl-Gly-Adc

[0104] 8 N-(BnSO2-norLeu(cyclo)-Gly-Adc

[0105] 93-({3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-2,4-dioxo-1,2,3,4-tetrahydro-quinazolin-6-carbonyl-amino)-propionicacid

[0106] 10 (4-3-2-(1-amidino-2,5-dihydro-1H-pyrro-1-yl)-ethyl]-2-oxo-oxazolidin-5-ylmethoxy}-phenyl)-acetic acid

[0107] 11.3-({1-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]-piperidin-3-carbonyl-amino)-3-pyridin-2-yl-propionic acid

[0108] 12.(4-{[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino]-acetyl-3-methoxycarbonylmethyl-piperazin-1-yl)-aceticacid

[0109] 13.[4-({[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-methylamino-acetyl)-phenoxyl-aceticacid

[0110] 14[7-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]-3-oxo-4-phenethyl-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-2-yl}-aceticacid

[0111] 153-(4-13-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-2-oxo-imidazolidin-1-yl}-phenyl)-propionicacid

[0112] 164-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-2-oxo-imidazolidin-1-yl}-cyclohexanecarboxylic acid

[0113] 17.[5-(4-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino]-methyl}-phenoxymethyl)-2-oxo-pyrrolidin-3-yl]-aceticacid

[0114] 18[5-({4-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino3-piperidin-1-yl}-acetyl)-2-carboxymethoxy-phenoxyl-aceticacid

[0115] 19(4-{[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-methyl-amino}-[1,4′]bipiperidinyl-1′-yl)-aceticacid

[0116] 20.3-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]-propionylamino}-butyricacid

[0117]21.3-{4-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]-propionylamino}-3-phenyl-propionicacid

[0118] 22.3-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]-propionylamino}-3-3 -pyridin-2-yl-propionic acid

[0119] 23.3-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylcarbamoyl]propionylamino{-pent-4-ynonecarboxylic acid

[0120] 243-(2-{3-[-2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-ureido-acetylamino)-N-(carboxy-phenyl-methyl)-succinicacid

[0121] 25.3-(2-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl}-2-oxo-tetrahydro-pyrimidin-1-yl}-acetylamino)-propionicacid

[0122] 26.{4-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino]-cyclohexyl-aceticacid

[0123] 273-(Butane-1-sulfonyl)-2-(4-{[2-(1-amidino-2,5-dihydro-1H-pyrrol-′)-yl)-ethylamino]-methyl}-benzyl)-propionic acid

[0124] 28.2-{2-[2-Amino-3-(1-amidino-2,5-dihydro-1H-pyrrol-3-yl)-propionylamino]-acetylaminol}-succinicacid

[0125] 29.1-{3-[2-(1-Amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-2-oxo-oxazolidin-5-ylmethyl}-piperidin-4-carboxylicacid

[0126] 30. Ada-Gly-Asp-Ser

[0127] 31. Ada-Gly-Asp-Ser-NH2

[0128] 32. Ada-Gly-Asp-Phe

[0129] 33. Ada-Gly-Asp-Phe-NH2

[0130] 34 Ada-Gly-Asp-Tyr

[0131] 35 Ada-Gly-Asp-Tyr-NH2

[0132] 36. Ada-Gly-Asp-Trp

[0133] 37. Ada-Gly-Asp-Trp-NH2

[0134] 38. Ada-Gly-Asp-Val

[0135] 39 Ada-Gly-Asp-Val-NH2

[0136] 40 Pla-Phe-Choi-Adc

EXAMPLE 1 D-Pla-D-Phe-L-Choi-Adc

[0137]

[0138] Production of the Biomass

[0139] Three 20 l glass cylinders with a pH regulator were filled withsterile filtered nutrient medium, each was inoculated with 50 mlpreculture of Oscillatoria agardhii from the collection of algaecultures of the Institute of Plant Physiology of the University ofGottingen (strain number B3 82) and incubated for ca 14 days at aconstant pH of 8 5, a temperature of 20° C. and an average irradiationof 30 Wm-2 under continuous gassing with sterile-filtered room air.

[0140] Nutrient Medium:

[0141] 3.6 mg CaCl2×2 H2O 20.3 mg Ca(NO3)2×4 H2O 2.7 mg KCl, 15 mgK2HPO4, 76.0 mg MgSO4×7 H2O 84.0 mg NaHCO3, 1 ml trace element solution,1000 ml deionized water.

[0142] Trace element solution: 875 mg Na4EDTA, 9 7 mg Co(NO3)2×6 H2O 284mg FeCl2×6 H2O 72.2 mg MnCl2×4 H2O 25.2 mg Na2MoO4×2 H2O 43.7 mg ZnSO4×7H2O, 1000 ml deionized water (K. Zielinski, Dissertation, Univ Freiburg,1988, p.23)

[0143] Processing of the 3×20 l cultures:

[0144] At the time of harvesting the three 20 l cultures were combinedand the cells and nutrient solution were separated by gentlecentrifugation (5000 g, 10 min). The biomass was frozen at −20° C. andsubsequently lyophilized.

[0145] The lyophilisate (ca. 30 g) was extracted once with 1000 ml andtwice with 500 ml methanol and the combined methanol phases wereevaporated to dryness. The methanol extract (ca. 7 g) was taken up in500 ml water and shaken out three times with 500 ml butanol each timeThe butanol phases were combined and concentrated to dryness at 40° C.on a rotary evaporator.

[0146] The butanol extracts were combined (ca. 5 g), taken up in 150 mlmethanol and absorbed onto ca. 25 g LiChroprep-CN phase (25-40 μm) andchromatographed over a LiChroprep-CN column (column: 52×356 mm; mobilephase gradient: water/acetonitrile/trifluoroacetic acid from (100:0:0:1)to (50:50:0.1), total elution volume 5 1).

[0147] The fractions containing D-Pla-D-Phe-L-Choi-Adc which wereidentified with the aid of TLC or the thrombin time prolongation assaywere pooled and evaporated to dryness at 40° C. on a rotary evaporator.

[0148] The concentrate (ca 1 g) was dissolved in 100 ml methanolabsorbed to ca. 5 g silica gel LiChroprep Si60 (15-25 μm) andchromatographed over a silica gel column (26×360 mm) with 1500 mlchloroform/methanol/glacial acetic acid/water (65.25 3 4) as the mobilesolvent

[0149] The pooled and concentrated fractions containingD-Pla-D-Phe-L-Choi-Adc (ca. 200 mg) were taken up in 3 ml methanol forthe further purification and further separated by high pressure liquidchromatography on Nucleosil-100 RP18 (10 μm, column 20×250 mm) withwater/acetonitrile/trifluoroacetic acid (70:30:01) as the mobile solvent

[0150] The concentrated fraction containing D-Pla-D-Phe-L-Choi-Adc (20mg) was taken up in 1 ml methanol and subjected to a further highpressure liquid chromatography (column: Nucleosil-100 RP18, 10 μm,20×250 mm; mobile solvent 50 mM phosphate buffer pH 7 8/ acetonitrile(70:30)). A fraction was obtained which only containedD-Pla-D-Phe-L-Choi-Adc. This was concentrated to ca. 5 ml aqueoussolution.

[0151] In order to remove the buffer the aqueous concentrate was appliedto a Nucleosil-100 RP 18 column (15×100 mm), wherebyD-Pla-D-Phe-L-Choi-Adc was bound to the stationary phase. After washingwith 100 ml water, the D-Pla-D-Phe-L-Choi-Adc was eluted with 200 mlwater/methanol (10:90). The methanolic eluate was evaporated to drynessat 40° C. on a rotary evaporator. ca. 3 mg pure D-Pla-D-Phe-L-Choi-Adcwas obtained as a colourless phosphate salt.

[0152] Analytical data for D-Pla-D-Phe-L-Choi-Adc:

[0153] a) Mass Spectrometry

[0154] The LSIMS spectrum of D-Pla-D-Phe-L-Choi-Adc yielded apseudomolecule ion at MH+ 617 Da. High resolution measurements in thepositive LSIMS mode resulted in MH+ 617.345 Da which enabled C34H44N6O5to be derived as the elemental composition of the neutral compound. TheMS/MS spectrum of the molecular ion MH+ produced a series of daughterions which confirmed the chemical structure derived forD-Pla-D-Phe-L-Choi-Adc.

[0155] Experimental Conditions:

[0156] The compound was measured at a resolution of ca 5000 in the LSIMSmode against PEG as the reference substance in the peak match mode.

[0157] Experimental Data

[0158] LSIMS selected peaks (M/Z.rel.int.) 136 92, 154 100, 176.9,193.6, 239 6, 289 14, 307:19: 331 3, 358.34, 381.2, 399. 6. 469.2,525:3, 593 7, 617 86, 647.8, 667:3, 713:6. MS/MS of MH+ selected peaks(M/Z:rel. int.) 94 10, 120 64. 140:39, 153.3, 1819.209 16;:250 2, 268.7.305:1. 320:13, 349.1; 377:3, 453:2, 469.16; 540.3; 565 5

[0159] Assignment of the fragments (daughter ions):

[0160] (b) Gas chromatographic analysis of the acid total hydrolysate ofD-Pla-D-Phe-L-Choi-Adc

[0161] The molecular components D-Pla and D-Phe could be detected in theacid total hydrolysate of D-Pla-D-Phe-L-Choi-Adc by gas chromatographicanalysis on a chiral phase. Furthermore a peak was identified in the gaschromatogram by means of GC-MS coupling which could be assigned in theEl mass spectrum to the molecular component Choi on the basis of the twotypical masses of 419 Da (the molecular ion (M+) ofN,O-di-trifluoroacetyl-Choi-n-propyl ester) and 332 Da (the fragment[M-C(O)OC3H]+)

[0162] Experimental Conditions

[0163] The sample was hydrolyzed for 24 h with 6 N hydrochloric acid andderivatized after blowing off the hydrochloric acid.

[0164] 0.5 ml 4 N hydrochloric acid in n-propanol was added to the dryhydrolysate for the detection of D-Phe and Choi and the reaction mixturewas heated for 30 minutes to 110° C. The excess reagent was completelyblown off The subsequent acylation was carried out. for 10 minutes at150° C. with trifluoroacetic acid anhydride. The reagent was againcompletely blown off and the sample was taken up in solvent. For thedetection of D-Pla 0.5 ml 4 N hydrochloric acid in methanol was added tothe dry hydrolysate and the reaction mixture was heated for 10 minutesto 110° C. The excess reagent was completely blown off. The subsequentaminolysis was carried out at room temperature with n-propylamine.Afterwards it was silylated for 20 minutes at 70° C. withhexamethyldisilazane.

[0165] The derivatized products of hydrolysis were analysed by capillarygas chromatography on a chiral phase. A 20 m capillary with an insidediameter of 0.28 mm and a film thickness of 0.25 μm was used which iscovered with Chirasil Val. A flame-ionization detector was used for thedetection. The peaks were assigned in the case of D-Phe and D-Pla bycomparing the retention with those of reference substances. In the caseof Choi a sector field mass spectrometer was used for the detection.

[0166] (c) NMR Spectroscopy

[0167] The structure of the compound of example i was elucidated andconfirmed by NMR spectroscopy (2D NMR, HMBC, HMQC, COSY (DQF-H,H-COSY,E,COSY), TOCSY, ROESY)

EXAMPLE 2

[0168] The compound of example 1 cocrystallizes with the bovine serineprotease trypsin. It was possible to derive the connectivity of theinhibitor in the complex with bovine trypsin from the high resolutioncrystal structure of the serine protease inhibitorD-Pla-D-Phe-L-Choi-Adc. Comparison with an arginine-containing inhibitorshowed that D-Pla-D-Phe-L-Choi-Adc contains an arginine mimetic

[0169] The inhibitor D-Pla-D-Phe-L-Choi-Adc resembles the known thrombininhibitor (D)-Phe-Pro-Arg (DFPR) along the main chain There aredifferences at the N-terminus (an additional residue), at the prolineanalogue (a condensed ring with a hydroxy group) and at the C-terminus(no carboxylate and a guanidino group partially integrated into afive-membered ring).

[0170] A comparison between the X-ray structure ofD-Pla-D-Phe-L-Choi-Adc with trypsin and that of the trombin-DFPR complex(Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S., & Hofsteenge, J.(1989). EMBO Journal 8, 3467-3475) clearly showed this similarity. Ifboth X-ray structures are superimposed, the DFPR inhibitor lies withinthe electron density of D-Pla-D-Phe-L-Choi-Adc. Deviations only occurwith respect to the differences in the side chain described above.

[0171] The Fo-Fc electron densities phased independently of the statedinhibitor structure confirmed the chemical structure of the molecularbuilding block Adc. The five-membered ring is obvious. The spatialgeometry of the electron density corresponds to a double bond in anequivalent position to the C of arginine. The planarity of the densityis also consistent with a nitrogen atom in the N equivalent position.From this the guanidino group is deduced.

[0172] The refinement of the X-ray structure of the inhibitorD-Pla-D-Phe-L-Choi-Adc with the protein structure—at first with theappropriate geometric restraints, subsequently without anyrestraints—showed that the molecular building block Adc ofD-Pla-D-Phe-L-Choi-Adc binds as an arginine analogue (FIG. 1). Theadditional ring atoms replace a water molecule The other specifichydrogen bridges as well as the spatial position of the correspondingatoms remain almost identical

[0173] Description of the Methods of X-ray Structural Analysis

[0174] Crystallization:

[0175] Bovine β-trypsin (SIGMA) was repurified (D. D. Schroeder, E. Shaw(1968) J Biol. Chem. 243, 2943-2949) and lyophilized. Forcrystallization preparations 3 5 mg of the lyophilisate (70% w/w betatrypsin, 20% benzamidine, 10% CaCl2) was dissolved in 30 μl distilledH20 and mixed 1: I with the precipitating solution (1.6 M ammoniumsulfate, 100 mM imidazole/H2SO4, pH 6, 0.02 % sodium azide). Crystalswere produced by vapour diffusion (6 Al drops protein solution/4 mlprecipitation solution) Benzamidine was replaced byD-Pla-D-Phe-L-Choi-Adc in the crystal by successive soaking: 1) twicefor 2 hours in a pure harvest solution (2 5 M ammonium sulfate, TRIS/HClpH 8. 10 mM CaCl2) in order to remove benzamidine and 2) overnight ininhibitor solution (1.76 mg D-Pla-D-Phe-L-Choi-Adc dissolved in 176 [11harvest solution).

[0176] Data Collection, Evaluation

[0177] The crystal was oriented with a 4-circle goniometer from Siemens.The X-ray source was a copper rotating anode Rigaku Rotaflex-generator(45 kV, 120 mA) equipped with a curved graphite monochromator. Thediffracted reflexes were measured with a Siemens X1000 area counter. Atotal of 2464 recordings (width 0.1 degree) of four orientations weremade corresponding to a theoretical completeness of 99% for a resolutionof 1.6 A (estimated with the program ASTRO (Siemens IndustrialAutomation, Inc., Madison, Wis., USA)). Automated indexing and dataevaluation were carried out using SADIE and SAINT (Siemens IndustrialAutomation. Inc., Madison. Wis. (see Table 1).

[0178] Table 1: Crystallographic Data and Refinement Statistics

[0179] Recording turning range: 0.1 degree

[0180] crystal detected removal: 11.65 cm recording time-90 sec.

[0181] total number of recordings: 541+541+541+841=2464

[0182] cell dimensions: 63.31 A, 63.57A. 69 06A, 90 deg, 90 deg. 90 deg.

[0183] spatial group- P2(1)2(1)2(1)

[0184] total reflexes. 65572

[0185] independent reflexes: 40648

[0186] positive reflexes. 34497

[0187] positive reflexes after 2 sigma selection: 28962

[0188] Total R-merge (5-1.5A): 4.2%

[0189] Completeness after resolution Resolution % in resolution shellTotal 2.90, 6.00 85.3264 85.3264 2.35, 2.90 95.2330 90.2261 206, 2.3591.3768 90.6049 1.88, 2.06 83.5322 88.8642 1.75, 1.88 68.6958 84.87841.65, 1.75 50.7991 79.2804 1.57, 1.65 28.7895 72.1611 1.50, 1.57.14.5349 65.0376

[0190] Model refinement

[0191] (p=protein, s=solvent, i=inhibitor,

[0192] r=rigid body refinement, c=Powell conjugate gradients refinement,t=B-factor

[0193] refinement, b=bonds, a=angles) Cycle ATOMS(p.s.i) %R-factor(r,c,t) RMS from ideality(b,a) 1 1630,23,0 28 7,27.2,23.40.007,1.97 2 1630,23,0 —, 22.8,22.5 0.006,1.72 3 1630,23,34 —, 21.7,21.50.007,1.74 4 1630,23,45 —, 21.5,21.4 0.006,1.80

[0194] Electron Density Calculation, Interpretation

[0195] The first electron density was phased with the 1.5 A resolvedP2(1)2(1)2(1) X-ray structure of trypsin (H. D. Bartunik, J. Summers, H.H. Bartsch, J. Mol. Bio. 210, 813, 1989) from the Brookhaven databank(ITLD, Abola, E. E. Bernstein, F. C., Bryant, S. H. Koetzle, T. F. &Weng, J. (1987) in Crystallographic databases—Information Content,Software Systems, Scientific Applications, Allen, F. H., Berghoff & G .Sievers, R., eds., Data Commission of the International Commission ofthe International Union of Crystllography (Bonn/Cambridge/Chester,1987), 107-132) after structural and B factor refinement using XPLOR(Molecular Simulations Incorporated MSI AG, Basel Switzerland). Thisdensity clearly showed the general orientation of the inhibitorAdditional refinement cycles improved the densities and thus enabled thedetermination of the connectivities of the basic group inD-Pla-D-Phe-L-Choi-Adc. It was also possible to likewise determine allother positions of the inhibitor except the first phenyl ring ofoscillarin which probably binds in a disordered manner

[0196] (a) 4-Oxo-pyrrolidine-1,3-dicarboxylic acid 1-tert. butylester3-ethylester

[0197] (J. Cooper et al. J. Chem. Soc. Perkin Trans. 1, 1993, 1313-1318)To a refluxing suspension of 1.58 g (66 mmol) sodium hydride in 100 mlTHF was added dropwise a solution of 12.79 g (60 mmol)N-tert-butyloxycarbonyl-glycine ethyl ester and 7.15 g (66 mmol) ethylacrylate in 100 ml THF After the addition was complete the mixture washeated to reflux for additional 2 h The clear solution was cooled toroom temperature, poured on 100 ml ether/100 ml water and acidifiedunder vigorous stirring with 1 N hydrochloric acid against methylorange. The layers were separated and the aqueous layer was extractedthree times with ether. The combined organic layers were washed withsat. sodium bicarbonate and brine, dried over MgSO₄ and evaporatedShort-path distillation of the residue gave 10 92 g (71%)4-oxo-pyrrolidine-1,3-dicarboxylic acid I-tert butylester 3-ethylesteras a colorless oil. b p. 119-122° C. (0.2 mbar). which solidified onprolonged standing in the freezer

[0198] GC/MS (HP 5890 It/HP 5972, column: HP 5, 30 m×25 mm×0 25 μm filmthickness, carrier gas helium; temperature gradient: 50° C. 3 min. thenwith 20° C./min to 250° C.) t_(R)=9.68 min m/z [%]- 185 (2), 130 (10)112(18), 85 (6), 57 (100)

[0199] b) 4-Hydroxy-pyrrolidine-1,3-dicarboxylic acid 1-tert.-butylester3-ethylester To a solution of 5.15 g (20 mmol)4-oxo-pyrrolidine-1,3-dicarboxylic acid 1-tert butylester 3-ethylesterin 30 ml methanol was added 1 88 g (30 mmol) sodium cyanoborohydride anda small amount of methylorange With stirring the pH was adjusted to 3 bydropwise addition of I N hydrochloric acid (color change from yellow toorange) After no more acid was consumed the mixture was stirred for onehour. The solvent was evaporated in vacuo and the residue waspartitioned between ethyl acetate and water The organic layer was washedtwice with water, then with brine, dried over magnesium sulfate andevaporated. The residual yellow oil was used in the next step withoutany further purification.

[0200] GC/MS (HP 5890 II/HP 5972, column: HP 5, 30 m×25 mm×0.25 μm filmthickness carrier gas helium; temperature gradient 50° C. 3 min: thenwith 20° C./min to 250° C.) t_(R)=12.44 min (no separation ofdiastereomers) m/z [%]=259 (M 0.3), 241 (0 7), 202 (5), 186 (7), 158(10), 112 (14), 68 (31), 57 (100).

[0201] c) 4-Benzoyloxy-pyrrolidine-1,3-dicarboxylic acid1-tert.-butylester 3-ethylester To an ice-cooled solution of the crude4-hydroxy-pyrrolidine-1,3-dicarboxylic acid 1-tert.-butylester3-ethylester from the reduction described above and 244 mg (2 mmol) DMAPin 40 ml pyridine were added dropwise 3.51 g (25 mmol) benzoyl chlorideAfter the addition was complete, the ice bath was removed and themixture was stirred at room temperature for 2 h. The mixture was dilutedwith ethyl acetate an poured on ice. The organic layer was separated,washed with water. sat. CuSO₄. water and brine, dried over MgSO₄ andevaporated. The residual yellow oil was used in the next step withoutfurther purification

[0202] GC/MS (HP 5890 l/HP 5972, column: HP 5, 30 m×25 mm×0.25 μm filmthickness, carrier gas: helium, temperature gradient: 50° C., 3 min,then with 20° C./min to 250° C.) t_(R)=17.28 and 17.38 min (1:1-mixtureof cis/trans-isomers) m/z [%] =318(0.1), 290(5), 262(2), 241 (2), 185(29), 141 (10), 112(23), 105 (53), 77 (27), 68 (100), 57 (97).

[0203] d) 2,5-Dihydro-pyrrole-1,3-dicarboxylic acid 1-tert-butylester3-ethyl ester

[0204] To a solution of the crude4-benzoyloxy-pyrrolidine-1,3-dicarboxylic acid 1-tert-butylester3-ethylester from the benzoylation described above in 75 ml dry toluenewas added 4.11 g (27 mmol) DBU. The dark, heterogeneous mixture wasstirred at room temperature for 16 h. After this time no startingmaterial was detectable by TLC and GC analysis The mixture was filteredthrough a short column of silica (elution with petrolether/ethyl acetate1 . 1) and evaporated. Bulb-to-bulb distillation of the residualslightly yellow oil gave 4 16 g (86%)2,5-dihydro-pyrrole-1,3-dicarboxylic acid 1-tert.-butylester 3-ethylester as a colorless oil b p. 110° C./0 2 mbar, which slowly solidifiedto a waxy mass on standing in the freezer.

[0205] GC/MS (HP 5890 II/HP 5972, column: HP 5, 30 m×25 mm×0.25 μm filmthickness, carrier gas helium, temperature gradient 50° C. 3 min, thenwith 20° C./min to 250 ° C.) t_(R)=11 94 min m/z [%]=241 (M , 1.4),196(0.4), 185(11), 168(11), 140(14), 112(17), 68(24), 57 (100).

[0206]¹H-NMR (CDCl₃300 MHz) δ=1.27 (t, J=7 1 Hz, 3H, OCH₂CH₃), 1.43,1.44 [2s, 9H, C(CH₃)₃]^(R), 4.25 (d, J=7.1 Hz, 2H, OCH₂CH₃), 4.15-4.27(br. m, 4H, 2-H, 5-H), 6.66-6.71 (m, 1 H, 4-H) ppm. “Double set ofsignals due to hindered rotation.

[0207]¹³C-NMR (CDCl₃75 MHz) δ=14.16, 14.20 (q, —CH₂—CH₃*, 28.45 [q,—C(CH₃)₃]51 76, 51 99, 53.62, 53 84 (4t, C-2. C-5)*. 60.69 (t,—CH₂—CH₃), 79 84 [s, —C(CH₃)₃],132.29 (s, C-3), 136 44, 136.55 (2d,C-4)*, 153.86, 15408 [(2s, —NCOO—)*, 162 75(s. COOEt) ppm

[0208] e) 3-Hydroxymethyl-2,5-dihydro-pyrrole-1-carboxylic acidtert.-butylester

[0209] To a solution of 5.43 g (22.5 mmol) 2.5-dihydro-pyrrole-1.3-dicarboxylic acid 1-tert.-butylester 3-ethyl ester in 50 ml THF,cooled to −78° C. was dropwise added 50 ml of a 1 N DIBAL-H solution inhexane. The mixture was allowed to warm to room temperature overnight.As TLC analysis indicated complete consumption of starting material, themixture was cooled in an ice bath and 1.90 g water were cautiouslyadded, followed by 1.90 g 15% aqueous NaOH and 5.70 g water. The whiteprecipitate was filtered off, washed thoroughly with ether and thecombined filtrates were evaporated. Bulb-to-bulb distillation of theresidual pale yellow oil gave 4 1 3 g (93%)3-hydroxymethyl-2,5-dihydro-pyrrole-1-carboxylic acid tert.-butylesteras a colorless oil, b.p. 130° C. (0.2 mbar).

[0210] GC/MS (HP 5890 II/HP 5972; column: HP 5, 30 m×25 mm×0.25 μm filmthickness, carrier gas: helium; temperature gradient: 50° C., 3 min;then with 20° C./min to 250° C.) t_(R)=11.34 min m/z [%]=199 (M, 1), 143(10), 142 (13), 126 (13), 11 2(12), 80 (10), 68 (45), 57 (100)

[0211]¹H-NMR (CDCl₃300 MHz) δ=1 44 (s, 9H, t-Bu), 4 09 (br. m. 4H, 2-H,4-H), 4.18 (br. s, 2H, CH₂OH), 5 63 (br d, 1H. 4-H) ppm ¹³C-NMR (CDCl₃75MHz) δ=28.5 [q, C(CH₃)₃], 52.8, 53 0, 53 2, 53 3 (4t, C-2, C-5), 57 7,59 8 (2d, CH₂OH)Fehler! Textmarke nicht definiert., 79.5 [s, C(CH₃)-],120.0, 120.3 (2d, C-4) Fehler! Textmarke nicht definiert., 139 6 (s,C-3), 154 4 (s, COOtBu) ppm. Double set of signals due to hinderedrotation

[0212] f) 3-Acetoxymethyl-2,5-dihydro-pyrrole-1-carboxylic acidtert-butylester To an ice-cooled solution of 4 13 g (20.7 mmol)3-hydroxymethyl-2,5-dihydropyrrole-1-carboxylic acid tert.-butylesterand 244 mg (2 mmol) DMAP in 50 ml pyridine was added 3 06 g (30 mmol)acetic anhydride The mixture was stirred for 30 min at 0° C., then foradditional 60 min at room temperature. The mixture was poured on ice andextracted twice with ether. The combined organic layers were evaporatedin vacuo, dissolved in ether, washed with sat. CuSO₄, water and brineand dried over MgSO₄. Evaporation and bulb-to-bulb distillation gave4.82 g (97 %) 3-acetoxy-methyl-2,5-dihydro-pyrrole-1-carboxylic acidtert.-butylester as a colorless oil, b.p. 105° C. (0.2 mbar).

[0213] GC/MS (HP 5890 II/HP 5972; column: HP 5, 30 m×25 mm×0 25 μm filmthickness, carrier gas- helium; temperature gradient: 50° C., 3 min;then with 20° C./min to 250° C.) t_(R)=11 87 min m/z [%]=241 (M .0 2),226 (0 1), 185 (5) 166 (5), 125 (18), 108 (3) 81(13) 80 (23), 57 (100)

[0214]¹H-NMR (CDCl₃, 300 MHz) δ=1.43, 1.44 [2s, 9H, C(CH₃)₃]*. 2.04,2.06 (2s, 3H, OOCCH₃)*, 4.05-4.12 (br m, 4H, 2-H, 5-H), 4.61 (br. d,J=5.7 Hz, 2H, CH₂0), 5.66-5.73 (br. m, 1H, 4-H) ppm.

[0215]¹³C-NMR (CDCl₃, 75 MHz) δ=20 7 (q, OOCCH₃), 28 4 [q. C(CH₃)₃], 530. 53.2. 53 3 (3t. 2-C. 5-C)*. 60 8 (t, CH₂OAc), 79 5 [s, C(CH₃)₃], 1234, 123 8 (2d, C-4)*, 134 5. 134,6 (2s, C-3), 154 1 (s, NCOO), 170.5 (s,OOCCH₃) ppm

[0216] g)2-(1-tert.-Butoxycarbonyl-2,5-dihydro-1H-pyrrol-3-yl-methyl)-malonicacid dimetylester

[0217] 5.28 g (40 mmol) dimethyl malonate were cautiously added to anice-cooled suspension of 864 mg (36 mmol) sodium hydride in 80 ml THF.The resulting clear solution was added to a solution of 4.82 g (20 mmol)3-acetoxymethyl-2,5-dihydro-pyrrole-1-carboxylic acid tert.-butylesterand 462 mg (0 4 mmol) Pd(PPh,)₄ in 40 ml THF and the mixture was heatedto reflux for 20 h. The reaction mixture was cooled to room temperature,diluted with ether and quenched with sat. NH₄Cl. The organic layer waswashed with sat. NH₄Cl and brine, dried over MgSO₄ and evaporated. Theresidue was purified by flash chromatography (ethyl acetate/petrol ether4:1 to 2: 1) and bulb-to-bulb distilled to yield 4.86 g (77%) of2-(1-tert.-butoxycarbonyl-2,5-dihydro-1H-pyrrol-3-yl-methyl)-malonicacid dimetylester as a colorless oil, b.p 130 ° C./0.2 mbar

[0218] GC/MS (HP 5890 I/HP 5972, column: HP 5, 30 m×25 mm×0.25 μm filmthickness, carrier gas: helium; temperature gradient: 50° C., 3 min;then with 20 ° C./min to 250° C.) t_(R)=14.07 min m/z [%]313 (M, 0.1),257 (27), 240 (5), 126 (35), 82 (59), 80 (38), 57 (100)

[0219]¹H-NMR (CDCl₃, 300 MHz) δ=1 43, 1 44 [2s, 9H, C(CH₃)₃]*, 2.68 (m,2H, 3—CH₂), 3 57 [t, J=6 6 Hz. 1H. CH(COOCH₃)₂]. 3 71, 3.72 [2s, 6H.CH(COOCH₃)₂], 3 97-4 10 (br m, 4H, 2-H, 5-H), 5 44 (br m, 1H, 4-H) ppm

[0220]¹³C-NMR (CDCl₃, 75 MHz)

[0221] δ=28.0, 28.1 (2t, 3-CH₂—)Fehler! Textmarke nicht definiert., 28.5[q, C(CH₃)₃], 50 1 [d, CH(COOMe)₂]52.7 (q, OCH₃), 53.1, 53 3, 54 5, 54.9(4t, C-2. C-5)*, 79 4 [s, C(CH₃)₃], 121.0 (d, C-4), 135 8 (s, C-3), 1541 (s, NCOO), 169 0 (s, COOMe) ppm

[0222] h) 3-(2-Methoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylicacid tert.butyl ester

[0223] To a solution of 4.52 g (14.4 mmol)2-(1-tert.-butoxycarbonyl-2,5-dihydro-1H-pyrrol-3-yl-methyl)-malonicacid idimethylester n 140 ml DMF were added 1 94 g (108 mmol) water and1 26 g(21.6 mmol) sodium chloride. The mixture was degassed and heatedto 150° C. for 30 h under an atmosphere of argon. After this period oftime no starting material could be detected by TLC. The mixture wascooled to room temperature, diluted with 200 ml ether and washed threetimes with water, then with-brine. The organic layer was dried overMgSO₄ and evaporated. Purification by bulb-to-bulb distillation gave3.38 g (92 %) of3-(2-methoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylic acid tert.butylester as a colorless oil, b.p. 130° C. (0.2 mbar)

[0224] GC/MS (HP 5890 II/HP 5972, column: HP 5, 30 m×25 mm×0.25 4m filmthickness, carrier gas: helium; temperature gradient: 50° C. 3 min; thenwith 20° C./min to 250° C.) t_(R)=12 63 min m/z [%] 255 (M+, 0.2), 199(48), 196 (2), 182 (10), 126 (26), 82 (54), 80 (32), 57 (100).

[0225]¹H-NMR (CDCl₃, 300 MHz) δ=13.2, 13.2 [2s, 9H, C(CH;).]*,2.30-2.35, 2.40-2.44 (2m, 2H each, —CH₂—CH₂—), 3.58 (s, 3H, OCH₃), 391-4.00 (m, 4H, 2-H, 5-H), 5.29-5.34 (m, 1H, 4-H) ppm ¹³C-NMR (CDCl₃, 75MHz)

[0226] δ=23.7, 23.8 (t, 3-CH₂)*, 28.2 [q, C(CH,)3], 31.7 (t, CH₂COOMe),51 4 (q, COOCH₃), 52.8, 53.1, 54.4, 54.7 (4t, C-2, C-5)*, 78.9 [s,C(CH-.)-,], 118 9 (d, C-4), 137.8 (s, C-3), 153 9 (s, NCOO), 172.8 (s,COOCH,) ppm

[0227]3-[2-(2,2,2-Trichlorethoxycarbonyl)-amino-ethyl]-2,5-dihydro-pyrrole-1-carboxylicacid tert. butyl ester

[0228] To a solution of 3 57 g (14 mmol)3-(2-methoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylic acid tert.butyl ester in 45 ml THF/methanol/water 3 1 I was added 1 18 g (28 mmol)LiOH*H₂O and the mixture was stirred for 1 h at room temperature. Thesolution was diluted with water and washed three times with ethylacetate. The aqueous layer was cooled in an ice bath and acidified with1 N hydrochloric acid against methyl orange The turbid mixture wasextracted three times with ether, the combined organic layers were driedover MgSO₄ and evaporated to yield 3 41 g of a colorless solid, whichturned dark on standing The crude acid was dissolved in 140 ml drytoluene and 4.06 g (14 mmol) diphenyl phosphoryl azide and 1.47 g (14.5mmol) triethylamine were added The mixture was heated to 80° C.overnight The solvent was removed in vacuo, the residual brown oil wasdissolved in 20 ml THF and 3 14 g (21 mmol) 2,2,2-trichloroethanol and asmall amount of cuprous chloride were added The mixture was heated toreflux for 2 h, then the solvent was evaporated and the residue waspurified by flash chromatography (petrol ether/ethyl acetate 3 I to 2.1)to yield 4 31 g (79 %)3-[2-(2,2,2-trichlorethoxycarbonyl)-amino-ethyl]-2,5-dihydro-pyrrole-1-carboxylicacid tert butyl ester as a colorless solid, m.p. 97-98° C.

[0229]¹H-NMR (CDCl₃, 200 MHz)

[0230] δ=1.41 [s, 9H, C(CH₃)₃]2.31 (br m, 2H, 3-CH₂—), 3.29-3.39 (m, 2H,CH₂NH), 4 67 (br. m, 4H, 2-H, 5-H), 4.67 (s, 2H. CH₂CCl₃), 5 43 (br m, IH. 4-H) ppm

[0231]¹³C-NMR (CDCl₃, 50 MHz)

[0232] δ=28.6 [q, C(CH₃)₃], 29.2 (t, —CH₂—CH₂—NH), 39.2 (t,—CH₂—CH₂—NH), 53.2, 53.4, 54.6, 54.9, (4t, C-2, C-5), 74.5 (d, CH₂CCl₃),79.5 [s, C(CH₃)₃]. 95.8 (s, CH₂CCl₃), 120 8. 121 0 (2d. C-4), 136 (s.C-′). 154.2, 154.6 (2s. NCOO) ppm

[0233] j)3-[2-(2,2,2-Trichlorethoxycarbonyl)-amino-ethyl]-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine

[0234] To an ice-cooled solution of 1.936 g (5 mmol)3-[2-(2,2,2-trichlorethoxycarbonyl)-amino-ethyl]-2,5-dihydro-pyrrole-1-carboxylicacid tert butyl ester in 10 ml dry dioxane was added I0 ml 4N hydrogenchloride in dioxane. The mixture was allowed to stand at 4° C. for 16 h.The solvent was then evaporated 1i7 vacuo without heating and thenevacuated in high vacuum The residue was suspended in 20 ml dryacetonitrile and 71 1 mg (5.5 mmol) ethyldiisopropylamine followed by1.614 mg (5 2 mmol)N,N-bis-tert.-butyloxycarbonyl-1H-pyrazole-1-carboxamidine were added.The clear solution was stirred for 2 h at room temperature, then thesolvent was distilled off and the residue was purified by flashchromatography (petrol ether/ethyl acetate 3 1 to 2:1 ) to yield 2.234 g(84 %) of a colorless solid, m.p. 121-123° C.

[0235]¹H-NMR (CDCl₃, 200 MHz)

[0236] δ=1 43 [s, 18H, C(CH₃)₃]2.27-2.33 (br m, 2H, 3-CH₂-), 3.28-3 38(m. 2H. CH₂NH), 4 31 (br. m, 4H, 2-H, 5-H), 4.65 (s, 2H, CH₂CC₃), 5.47(br. m, 1H, 4-H) pm.

[0237]¹³C-NMR (CDCl₃, 50 MHz) δ=28.0, 28.1 [2q, C(CH₃)₃], 29.0 (t,3-CH₂), 39.0 (t, CH₂NH₂), 55.4, 56.0 (2 br. t, C-2. C-S), 74 5 (d,CH₂CCl₃), 79.5, 82.0 [2s, C(CH₃)₃], 95.6 (s, CH2CC]₃), 119 9 (d, C-4),135.3(s, C-3), 150 4, 154.0, 154.5, 162.5(4s, NCOO, N=C-N) ppm.

[0238] k) 3-(2-Amino-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert-butoxycarbonyl) carboxamidine

[0239] To a solution of 2.234 g (4.22 mmol)3-[2-(2,2,2-trichlorethoxycarbonyl)-amino-ethyl]-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine in 20 ml glacial acetic acid was added 1.3 1 g (40 g-atom)activated zinc and the mixture was stirred at room temperature for 2 h.Methanol was added, the mixture was filtered through celite, washedthoroughly with methanol and the filtrate was concentrated in vacuo Theresidue was dissolved in water and made strongly alkaline with solidsodium hydroxide. The aqueous layer was extracted three times withether, the combined organic layers were dried over MgSO₄ and evaporatedto yield 622 mg (41%)3-(2-amino-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine as a slightly yellow, amorphous solid.

[0240]¹H-NMR (CDCl₃, 300 MHz)

[0241] δ=1.45 [s, 18H, C(CH₃)₃], 2.21-2.26, (m, 2H, 3-CH₂-), 2.83 (t,J=6.9 Hz, 2H, -CH₂-NH2), 4.28-4.34 (br. m, 4H, 2-H, 5-H), 5.46 (br. s,1H, 4-H) ppm. ¹³C-NMR (CDCl₃, 75 MHz) δ=27.85, 27.94 [2q, C(CH₃)₃], 32.2(t, 3-CH₂), 39.5 (t, CH₂NH₂), 55.2, 56.7 (2t, C-2, C -5), ca 81 [br. s,C(CH-)-], 119.0 (d, C-4), 136.1 (s, C-3), 153.7 (s, NCOO) ppm.

[0242] l) HOOC—CH₂-D-Cha-(N-cyclopentyl)-Gly-Adc

[0243] A mixture of 0.41 g (0.81 mmol)N-(tert.butyloxycarbonyl)-N-(tert.butyloxycarbonyl-methyl)-(D)-cyclohexylalanyl-N-cyclopentylglycine,0.15 ml (0.9 mmol) diisopropylmethylamine, and 0.29 g (0.9 mmol) TBTU in25 ml dry DMF was stirred at room temperature for 30 min. Then 0.3 g(0.85 mmol)(3-(2-amino-ethyl)-2,5-dihydropyrrole-(1-(N,N′-di-tert.butoxycarbonyl)-carboxamidinewas added and strirred for 70 h. The solvent was removed i.vac., waterwas added and extracted with ethyl acetate The ethyl acetate wasseparated, washed with 1 M hydrochloric acid and 5% aqueous sodiumbicarbonate and dried over sodium sulfate. Filtration and removal of thesolvent i. vac produced 0.35 g (51%) N-(2-{[1-(N,N′-di-tertbutoxycarbonyl)-carboxamidino]-2,5-dihydro-pyrrol-3-yl-ethyl)-[N-(tert.butyloxycarbonyl)-N-(tert.butyloxycarbonyl-methyl)-D-cyclohexylalanyl-(N-cyclopentylglycin)]amideas a colorless oil. FAB-MS: m/z 847 MH

[0244] A solution of the above product (0.35 g, 0 41 mmol) in 10 ml 4 Nhydrogen chloride in dioxane was stored for 24 h at 5° C. The solventwas removed i.vac, and the oily residue was triturated with diethylether to yield the title compound as hydochloride quantitatively.FAB-MS: m/z 491 M

[0245]¹H-NMR (d₆-DMSO, 250 MHz). δ=0 8-4 8 ppm (m broad). 5 68 (s. 1)

[0246] The title compound was synthesized as hydrochloride fromBoc-N-Me-D-Phe-ProOH (Bajusz et al. J. Med. Chem 1990, 33, 1729-1735)and 3-(2-amino-ethyl)-2,5-dihydropyrrole-1-(N,N-di-tert.-butoxycarbonyl) carboxamidine (example 3k) by the same methodas described in example 31) FAB-MS- m/z 412 MH

[0247] The title compound was synthesized as hydrochloride fromN-(tert.-butyloxycarbonylmethyl)-N-Boc-D-Cha-ProOH and3-(2-amino-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-Butoxycarbonyl)carboxamidine (example 3k) by the same method as described in example31) FAB-MS: m/z 463 MH

EXAMPLE 6

[0248]1-{3-[2-(1-amidino-2,5-dihydro-1H-pyrrol-3-yl)-ethyl]-2-oxo-oxazolidin-5-ylmethyl}-piperidine-4-carboxylicacid

[0249] (a) A mixture of 3 1 g piperidine-4-carboxylic acid ethyl ester,6 4 ml epichlorohydrin and 0 1 g tetrabutyl-ammonium bromide in 15 mltoluene and 15 ml concentrated sodium hydroxide solution was stirred for4 h at room temperature and subsequently admixed with 50 ml water. Theorganic phase was separated, the aqueous phase was shaken three timeswith 20 ml methylene chloride each time, the combined organic phaseswere dried over sodium sulfate and the solvent was removed in a vacuum.2.1 g (rac)-1-oxiran-2-ylmethylpiperidine-4-carboxylic acid ethyl esterwas obtained. El-MS: m/z 213M′.

[0250] (b) A solution of 3 g of the amine prepared in example 3k) and1.73 g of the oxirane produced in 7a) in 20 ml ethanol was heated for 48h under reflux. Subsequently the ethanol was removed in a vacuum and theresidue was purified by column chromatography on silica gel (ethylacetate/ saturated methanolic ammonia 85/15). 1.5 g1-{3-[2-(1-(N,N′-di-tert.-butoxycarbonyl)carboxamidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino]-2-hydroxy-propyl}-piperidine-4-carboxylicacid ethyl ester was obtained in this way

[0251] (c) A solution of 0 5 g of the aminoalcohol 7b) and 0.4 gcarbonyldiimidazole in 5 ml dimethylformamide was stirred for 24 h atroom temperature. Subsequently the reaction solution was concentrated todryness by evaporation and the residue was purified by means ofpreparative HPLC (Merck, Select B, methanol/buffer (pH=7.5) 65/35). 0.35g 1-{3-[2-(1-(N,N′-di-tert-butoxycarbonyl)carboxamidino-2,5-dihydro-1H-pyrrol-3-yl)-ethylamino]-2-hydroxy-propyl-piperidine-4-carboxylicacid ethyl ester was obtained in this manner

[0252] (d) A solution of 0.35 g of the ethyl ester 6c) and 2 ml 1Nsodium hydroxide solution in 5 ml methanol was stirred for I h at roomtemperature. Subsequently the methanol was removed in a vacuum and ttheresidue was trearted with 4M Hcl in dioxane for 3 hours at roomtemperature. Then the solvent was removed until drying in vacuo. 0.2 gof the title compound was obtained as hydrochlioride. FAB-MS. m/z366[MH].

EXAMPLE 7 Troc-Ada-Gly-Asp-Ser

[0253] a)3-(2-Benzhydrilideneamino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylicacid tert.-butylester

[0254] To a solution of lithium hexamethyldisilazide, freshly preparedat 0° C. from 1.77 g (11 mmol) hexamethyldisilazane in 10 ml THF and 480 g (11 mmol) n-Butyl-lithium. (2.29 mmol/g in hexanes) and cooled to−78° C. was dropwise added a solution of 3.06 g (11 mmol) ethylN-(diphenylmethylene)-glycinate in 10 ml THF. The deep orange enolatesolution was stirred at this temperature for 30 min, then a solution of2.41 g (10 mmol) 3-acetoxymethyl-2,5-dihydro-pyrrole-1-carboxylic acidtert-butylester from example 3f) and 426 mg (0.4 mmol) Pd(PPh.)₄ in 10ml THF was added dropwise. The mixture was allowed to warm to roomtemperature overnight and was then diluted with ether and quenched byaddition of sat. sodium bicarbonate The aqueous layer was extracted withether, the combined organic layers were washed with brine. dried overMgSO₄ and evaporated. Flash chromatography of the residual oil (elutionwith petrol ether/ethyl acetate 3.1+1% triethylamine) gave 3 91 g (81 %)3-(2-benzhydrilidene-amino-2-ethoxycarbonyl-ethyl-2,5-dihydropyrrole-1-carboxylicacid tert.-butylester as a slightly yellow oil.

[0255]¹H-NMR (CDCl3, 300 MHz)

[0256] δ=127 (t, J=7.1 Hz, 3H. OCH₂CH₃), 1 43 [s. 9H. C(CH₃)₃1.2.66-2.76 (m, 2H. 3-CH-), 373 -3 99(m, I H, CH-N). 4 04-4 09 (br. m. 4H.2-H, 5-H), 4 17 (q, J=7 1 Hz. 2H, OCH2CH3), 5 42 (br. m, 1H, 4-H), 711-7.81 (m, 10 H. Ar-H) ppm.

[0257]¹³C-NMR (CDC13, 75 Mhz) δ=14.2 (q, OCH2CH₃), 28.5 [q, C(CH₃)₃],33.1 (t, 3-CH₂), 52.9, 55.1 (2t, C-2, C-5), 61 1 (t, OCH2CH.), 63 9 (d,CH-NH2), 79 1 [s, C(CH-)₃], 121.9 (d, C-4), 127.7, 128.1, 128.3, 128.4,128.5, 128.7 (6d, Ar-CH), 135.7, 135.8 (2s, Ar-C), 139.3 (s, C-3), 154 1(s, NCOO), 170 8 (s, N=CPh₂), 171.2 (s, OCOCH₂CH₃) ppm

[0258] b)3-(2-Amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylic acidtert.-butylester

[0259] To 25 ml of a 0.5 M solution of methoxylamine hydrochloride in80% ethanol was added a solution of 448 mg (1 mmol)3-(2-benzhydrilideneamino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylicacid tert. -butylester in 5 ml chloroform. After 5 min TLC analysisindicated the complete consumption of the starting material. The mixturewas stirred for additional 30 min and then evaporated to dryness. Theresidue was dissolved in water and washed twice with ether. The aqueouslayer was made alkaline by addition of 1 N potassium hydroxide andextracted three times with ether The combined organic extracts weredried over MgSO₄ and evaporated to yield 214 mg (75%)3-(2-amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-carboxylic acidtert.-butylester as a colorless oil which was pure according to GC

[0260] GC/MS (HP 5890 II/HP 5972; column: HP 5, 30 m×25 mm×0.25 μm filmthickness, carrier gas helium; temperature gradient: 50° C., 3 min, thenwith 20° C./min to 250° C.) t_(R)=13.84 min m/z [%]=227 (M - C₄H₉, <1),211 (7), 182 (10), 155 (11), 137 (6), 126 (56), 108 (18), 94 (23), 82(100), 74 (20), 57 (92).

[0261]¹H-NMR (CDC13, 300 MHz) δ=1.23 (t, J=7.1 Hz, 3H, OCH₂CH—), 1.42[s, 9H, C(CH₃)3], 2.35 (dd, 21J 14.2 Hz, 3J=8 Hz, 1H, CH_(a)H_(b)CHNH₂),2.50-2.55 (m, 1H, CH_(a)H_(b)CHNH₂), 3 54 (dd, 3J =8, 5.5 Hz, 1H,CHNH₂), 4.02-4.11 (br. m, 4H. 2-H, 5-H), 4 13 (q, J =7 1 Hz, 2H, OCHCH₃), 5 51 (br. m, 1H, 4-H) ppm

[0262]¹³C-NMR (CDC13, 75 MHz) δ=14.1 (q, OCH₂CH₃), 28.5 [q, C(CH₃)₃], 345 (t, 3-CH₂), 52.9 (d, CH-NH₂). 53 0, 54.8 (2t, C-2, C-5), 61.0 (t,OCH₂CH₃), 79.3 [s, C(CH₃)₃], 122.2 (d, C4), 135.5 (s, C-3). 154 1 (s.NCOO), 175 0 (s, OCOCH₂CH;) ppm

[0263] c)3-[2-(2,2,2-Trichloroethoxycarbonyl-amino)-2-ethoxycarbonyl-ethyl]-2,5-di-hydro-pyrrole-1-carboxylicacid tert-butylester

[0264] To an ice-cooled solution of 214 mg (0.75 mmol)3-(2-amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole- -carboxylic acidtert. -butylester and 119 mg (1 5 mmol) pyridine in 2 ml methylenechloride was added a small amount of DMAP followed by 212 mg (1 mmol)2,2,2-trichloroethoxycarbonyl chloride. The mixture was allowed to warmto room temperature overnight. The mixture was diluted with ether,washed with water, sat. CuSO₄, water and brine, dried over MgSO₄ andevaporated. Purification of the residue by flash-chromatography (elutionwith petrol ether/ethyl acetate 3.1) gave 320 mg (93%)3-[2-(2,2,2-trichloroethoxycarbonyl-amino)-2-ethoxycarbonyl-ethyl]-2,5-dihydropyrrole-1-carboxylicacid tert.-butylester as a colorless oil, which was immediately used forthe next step

[0265] d)3-[2-(2,2,2-Trichloroethoxycarbonyl-amino)-2-ethoxycarbonyl-ethyl]-2,5-di-hydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine

[0266] To an ice-cooled solution of 320 mg (0.67 mmol)3-[2-(2,2,2-trichloroethoxycarbonyl-amino)-2-ethoxycarbonyl-ethyl]-2,5-dihydropyrrole-1-carboxylicacid tert.-butylester in 1.5 ml dry dioxane was added 1.5 ml 4 Nhydrogen chloride in dioxane. The mixture was allowed to stand at 4° C.for 16 h. The solvent was then evaporated in vacuo without heating. Theresidue was suspended in 4 ml dry acetonitrile and 217 mg (0 7 mmol)ethyldiisopropylamine followed by 97 mg (0 75 mmol)N,N′-bis-tert-butyloxycarbonyl-1H-pyrazole-1-carboxamidine were addedThe resulting clear solution was stirred for 2 h at room temperature,then the solvent was distilled off and the residue was purified by flashchromatography (petrol ether/ethyl acetate 3.1 to 2:1 ) to yield 380 mg(94 %) of a colorless foam

[0267]¹H-NMR (CDC13, 200 MHz) δ=1 21 (t. J=7.0 Hz. 3H, OCH₂CH₃). 1 43[s, 9H, C(CH₁)₃1. 2.53-2 64 (m. 21i. CH₂CHNH), 4 16 (q, J=7 0 Hz. 2H.OCH.CH₃). 4.29 (br. m, 4H, 2-H, 5-H), 4 67 s, 2H, CH₂CCl₃), 5.52-5 61(m, 3H, -CHNH,4-H) ppm. ¹³C-NMR (CDCI3, 50 MHz) 6=14 I (q, OCH₂CHI), 280. 28 2 [²q, C(CH₃)₃], 31.8 (t, 3-CHL), 52 9 (d, CH—NH₂), 55 4, 57.1(2br. t, C-2. C-S), 61.9 (t, OCH₂CH;), 74 7 (t, CH,CCI)), 79 5, 83 5[2s, C(CH₃)₃1, 95 3 (s, CH₂CCI₃), 121 9 (d, C-4). 133.3 (s, C-3), 154 0(s, NCOO). 171.1 (s, COOEt) ppm. (BOC-NCOO- and amidine -N-C=N-signalsnot visible due to line broadening).

[0268] e)3-12-(2-2,2-Trichloroethoxycarbonyl-amino)-2-carboxy-ethyl]-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl) carboxamidine To a solution of219 mg (0.36 mmol)3-[2-(2,2,2-trichloroethoxycarbonyl-amino)-2-ethoxycarbonyl-ethyl]-2,5-dihydropyrrole-1-(N,N′-di-tert-butoxycarbonyl) carboxamidine in 4 ml THF/methanol/water3:1:1 was added30 mg (0.72 mmol) LiOH*H2O. After stirring for 1 h at room temperatureno starting material could be detected by TLC. The mixture was dilutedwith water, acidified by addition of 1 N hydrogen chloride and extractedthree times with ethyl acetate. The combined organic extracts were driedover MgSO₄ and evaporated. The residue was purified byflash-chromatography (elution with ether+1% acetic acid) to yield 156 mg(76%) 3-[2-(2,2,2-trichloroethoxycarbonyl-amino)-2-carboxy-ethyl]-2,5-dihydropyrrole-1-(N,N′-di-tert-butoxycarbonyl)carboxamidine (Troc-Ada(Boc₂)-OH) as a colorless amorphous solid.

[0269]¹H-NMR (CDC13 250 MHz)

[0270] δ=1 42 [s, 18H, C(CH3)3], 2.58-2 79 (br m, 2H, CH₂CHNH). 4 30-438 (m. 4H. 2-H, 5-H), 4 58 (d, J=12.2 Hz, 1H, CH_(a)H_(b)CCl₃), 4.75 (d,J=12.2 Hz, 1H, CH_(a)H_(b)CCl₃), 5 53 (br. s, 1H, CHNH), 5 99 (br. m,1H, 4-H) ppm

[0271] f) Troc-Ada-Gly-Asp-Ser

[0272] The title compound was synthesized by solid-phase methodology ona SyRo 11 multiple peptide synthesizer (MultiSynTech, Bochum) on a 0.03mmol scale using Fmoc-Ser(t-Bu)-trityl-polystyrene(1%)divinylbenzeneresin (Fmoc-L-Ser(t-Bu)-TCP, loading: 0.57 mmol/g, PepChem, Tübingen) asstarting material. The α-amino groups of the proteinogenic amino acidsGly and Asp were protected by 9-fluorenylmethoxycarbonyl (Fmoc), theside chain carboxy group of Asp by tert.-butyl The non-proteinogenicamino acid Ada was used as Troc-Ada(Boc,)-OH (from example 8e). TheFmoc-protected amino acids were coupled in a 6-fold excess for 30 min inDMF TBTU (1 eq) and NNM (1 eq) were used as activating reagents Cleavageof the Fmoc group was carried out in piperidine/dimethylformamide (T. Iv/v) for 2×10 min Coupling of Troc-Ada(Boc,)-OH was performed manuallyin DMF within 1 h by using 0 048 mmol of the protected amino acid (I65-fold excess) and equimolare amounts of TBTU and NMM for activationThe peptide was cleaved from the resin, with 750 ul of aceticacid/trifluoroethanol/dichloromethane (30 10 70) within 2 h. Afterwashing five times with 150 ul of the same solvent mixture the filtrateswere combined, diluted with 10 ml heptane and concentrated. Thisprocedure was repeated twice in order to remove the acetic acidcompletely The oily residue was dissolved in 5 ml 4 N hydrogen chloridein dioxane. To this solution 270 ul ethanedithiol were added and themixture was stirred for 3 h at room temperature. Then the solvent wasremoved and the residue dissolved in heptane and concentrated againseveral times until the ethanedithiol was almost completely removed. Thecrude peptide was lyophilized from tert -butanol/water (1 1) and yielded15 mg of Troc-Ada-Gly-Asp-Ser.HCl as white lyophilisate.

[0273] Amino acid analysis: Gly 1.09(1), Asp 1.00(1), Ser 0.95(1),peptide content: 69 9% ESI-MS: m/z 631.1 M⁺

EXAMPLE 8 Troc-Ada-Gly-Asp-Trp

[0274] The title peptide was prepared in the same manner as example 7f)starting from 50 mg (0 03 mmol) Fmoc-L-Trp-TCP resin. 16 mg ofTroc-Ada-Gly-Asp-Trp·HCl were obtained as white lyophilisate.

[0275] Amino acid analysis: Gly 1.19 (1); Asp 0.88 (1); Trp 1.00 (1);peptide content 61.2% ESI-MS: m/z 730 2 M⁺

EXAMPLE 9 Troc-Ada-Gly-Asp-Phe

[0276] The title peptide was prepared in the same manner as example 7f)starting from 50 mg (0.0) mmol) Fmoc-L-Phe-TCP resin. 14 mg ofTroc-Ada-Gly-Asp-Phe * HCl were obtained as white lyophilisate

[0277] Amino acid analysis: Gly 1 08 (1): Asp 1 00 (1); Phe 0 93 (1),peptide content 65 0% pos. LSIMS: m/z 692.1 MH⁺

[0278] The Troc protecting group of the compounds of example 7 to 9 canbe removed by standard chemical reactions.

EXAMPLE 10 Ada-Gly-Asp-Tyr

[0279] a)3-Acetoxymethyl-2,5-dihydro-pyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine

[0280] To an ice-cooled solution of 1.21 g (5 mmol)3-acetoxymethyl-2,5-dihydro-pyrrole-1-carboxylic acid tert.-butylesterfrom example 3f) in 10 ml dry dioxane was added 10 ml 4N hydrogenchloride in dioxane. The mixture was stirred at 0° C. for 16 h Themixture was evaporated to dryness without heating and then evacuated inhigh vacuum for several hours. The dark residue was suspended in 20 mldry acetonitrile and 776 mg (6 mmol) ethyl diisopropylamine, followed by1 71 g (5.5 mmol) N,N′-bis-tert-butyloxycarbonyl-1H-pyrazole-1-carboxamidine were added. The mixturewas stirred for 2 h at room temperature and then evaporated and purifiedby flash chromatography (petrol ether/ethyl acetate 3.1 to 2:1) to yield1.87 g (97 %)of3-acetoxymethyl-2,5-dihydro-pyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine as a colorless, sticky solid

[0281]¹H-NMR (CDCl₃, 300 MHz) δ=1 45 (s, 18H. 2 t-Bu). 2.03(s. 3H. OAc),4 38 (br. m, 4H. 2-H. 5-H). 4 61 (s. 2H. CH₂OAc), 5.72 (br. m, 1H, 4-H),10.22 (br. s. 1H, NH) ppm

[0282]¹³C-NMR (CDCl₃, 75 MHz) δ=20 4 (q. OOCCH₃). 27 7. 7 9 [2q,C(CH.₃)₃], 5S 0 (br t. C-2, C-S). 60 2 (t. CH₂OAc), 79 3, 81.8 [2 br. s.C(CH₃)., 122.4 (d. C4), 133. 5(s. C-3). 150 (br s. NCOO), 153.9 (s,NC=N), 162 (br. s, NCOO), 170.2 (s, OOCH₂CH₃) ppm.

[0283] b)3-(2-Benzhydrilideneamino-2-ethoxycarbonyl-ethyl)-2.5-dihydropyrrole-1-(N,N-di-tert.-butoxycarbonyl) carboxamidine

[0284] To a solution of lithium hexamethyldisilazide, freshly preparedat 0° C. from 710 mg (4 4 mmol) hexamethyldisilazane in 8 ml THF and 192 g (4 4 mmol) n-Butyl-lithium. (2.29 mmol/g in hexanes) and cooled to−78° C. was added a solution of 1 069 g (4 mmol) ethylN-(diphenylmethylene)-glycinate in 8 ml THF The orange enolate solutionwas stirred for 30 min at −78° C., then a solution of 1 039 g (3 7 mmol)3-acetoxymethyl-2,5-dihydro-pyrrole-1-(N,N -di-tert. -butoxycarbonyl)carboxamidine and 426 mg (0 4 mmol) Pd(PPh₃)₄ in 12 ml THF was addeddropwise The reaction mixture was allowed to warm to room temperatureover 2 h and was stirred for additional 12 h. The mixture was dilutedwith ether and quenched by addition of sat NaHCO; The organic layer waswashed with sat. NaHCO; and brine, dried over MgSO₄ and .evaporated.Purification by flash chromatography (ethyl acetate/petrol ether 1 5+1%triethylamine) gave 1.03 g (47%) of3-(2-Benzhydrilideneamino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N-di-tert.-Butoxycarbonyl) carboxamidine as a colorless, amorphous solid.

[0285]¹H-NMR (CDCl₃, 300 MHz) δ=1 23 (t J=7 Hz. 3H. OCH₂CH₃), 1 46 [brs. 18H. C(CH₃)₃]2 68 (br m, 2H. 3—CH₂—), 3 96 (br m, 1H, CH-N), 4 15 (q.J=7 1 Hz, 2H, OCH2CH), 4 16-4.29 (br m, 4H, 2-H, 5-H). 541 (br m, 1H,4-H), 7 07-7 00 (m. 10 H. Ar-H) ppm

[0286]¹³C-NMR (CDCl₃, 75 MHz) δ=13.9 (q, OCH₂CH₃). 28 0 [q, C(CH₃)₃],32.5 (t, 3-CH₂), 55.2, 56 9 (2t. C-2. C-5). 60.9 (t, OCH₂CH₃), 63.6 (d,CH-NH₂), 79, 81.6 [2 br. s, C(CH₃)i], 120.7 (d. CA). 1275, 1278 1284,128.5. 1286, 130.2 (6d. Ar-CH), 1348(s, C-)), 135.8 1 39 I (2s. Ar-C ).I 0 (br s. NCOO). 153 7 (s. NC=N). 162 (br. s. NCOO). 170 7 (s, N=CPh₂),171 2 (s, OOCH₂CH₃) ppm.

[0287] c)3-(2-Amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine

[0288] To a solution of 118 mg (0.2 mmol)3-(2-benzhydrilideneamino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine in 2 ml THF was added 1 ml 1 N hydrochloric acid. Themixture was stirred at room temperature for 30 min. Water (5 ml) wasadded, the aqueous layer was separated and washed twice with ether. Theaqueous layer was brought to pH=8.5 by addition of 1N NaHCO₃ and wasextracted five times with ether The combined ether layers were washedwith brine, dried over MgSO₄ and evaporated. The residue was purified byflash chromatography (chloroform/methanol 20-1) to yield 79 mg (93%)3-(2-amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine as a colorless oil

[0289]¹H-NMR (CDCl₃, 300 MHz)

[0290] δ=1.22 (t, J - 7.1 Hz, 311, OCH₂CHt), 1.45 [s, 18H, C(CH₃)₃1,2.33 (dd, ²J=16.6 Hz, J=8-1 Hz, 1H, CH₃H₂CH₂),2.54 (dd, 2J=16 6 Hz,3.1=5 3 Hz, I H, CH_(a)H_(b)CHNH₂), 3.54 (dd, ³J=8. 1, 5.3 Hz, 1H,CH_(a)H₂), 4. I-3 (q,.J=7 1 Hz, 2H, OCH₂CH₃), 4.33 (br. m, 4H, 2-H,5-H), 5.53 (br. m, 1 H. 4-H) ppm

[0291]¹³C-NMR (CDCl₃, 75 MHz) δ=13 9 (q. OCH2CH₃). 27 9 [q, C(CH₃)₃], 341 (t. 3-CH₂). 52 7 (d. CHNH₂, 553. 56 9 (2d . C-2. C-5) 61.1 (t.OCH₂CH₃). ca 80 [2 br s, C(CH₃)₃]. 120 7 (d. C-4). 134 6 (s, C-3), 153 8(s, NC=N), 174.6 (s, OOCH₂CH₃) ppm.

[0292] d) 3-(2-tert.-Butoxycarbonyl-amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N ′-di-tert.-butoxycarbonyl) carboxamidine

[0293] To a solution or 79 mg (0.19 mmol)3-(2-amino-2-ethoxycarbonyl-ethyl)-2.5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl)carboxamidine in 1 ml dry acetonitrile was added 40 mg (0.3 mmol) ethyldiisopropylamine and 65 mg (0 3 mmol) di-tert.-butyl dicarbonate (Boc₂O)and the mixture was stirred for 16 h at room temperature. The solventwas evaporated and the residue was purified by flash chromatography(petrol ether/ethyl acetate 2 1) to yield 83 mSg (76 %) 3-(2-tert-butoxycarbonyl-amino-2-ethoxycarbonyl-ethyl)-2,5-dihydropyrrole-1(N,N-di-tert.-butuxycarbonyl) carboxamidine as acolorless oil.

[0294]¹H-NMR (CDCl₃, 200 MHz) δ=1.22 (t, J=7 1 Hz, 3H. OCH₂CH3), 1.42,1.47 1.48 [3 s, 9H each. C(CH₃))], 2.41-2.66 (br. m, 2H, 3-CH₂-), 4. 17(q, J=7.1 Hz, 2H, OCH₂CH.), 4.32 (br. m, 4H, 2-H, 5-H), 5.02 (br. m. 1H,CHNH), 5.52 (br s. 1H, 4-H) ppm

[0295]¹³C-NMR (CDCl₃, 50 MHz) δ−14.1 (q. OCHCH₃), 28 1. 28.3, 28.5 [3q,C(C.H₃)₃1, 31 8 (t. 3-CH.), 48 3 (d. (CHNH), 52 0, 5 3 (2t, (′-2. C-5),61.6 (t. OCH.CH₃), 121 3 (d. (′-4), 133 5 (s, C′-3), 153 9(s, N=C-N).171 8(s, COOEt) ppm NCOO—, C(CH₃)₃-signals not visible due to linebroadening

[0296] e)3-(2-tert.-Butoxycarbonyl-amino-2-carboxy-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl) carboxamidine

[0297] To a solution of 267 mg (0.63 mmol)3-(2-tert.-butoxycarbonyl-amino-2-ethoxy-carbonyl-ethyl)-2,5-dihydropyrrole-1-(N,N′-di-tert.-butoxycarbonyl) carboxamidine in 5 ml THF/methanol/water 3 1 1 wasadded 50 mg (1.2 mmol) LIOH*H₂0. After 30 min stirring at roomtemperature no starting material could be detected by TL-C The mixturewas made acidic by addition of 1 N HCl, diluted with water and extractedthree times with ether. The combined organic extracts were washed withbrine, dried over MgSO₄ and evaporated. The residue was purified byflash-chromatography to give 98 mg, (31 %) of3-(2-tert.-Butoxycarbonyl-amino-2-carboxy-ethyl)-2.5-dihydropyrrole-1-(N,N-di-tert.-butoxycarbonyl)carboxamidine (Boc-Ada( Boc.)-OH) as a colorless amorphous solid.

[0298]¹H-NMR (CDC13, 300 MHz)

[0299] δ=1.39, 1 44 [2s, 9H, 1i8H, C(CH))₃], 2.49-2.67 (br. m. 2H.3-CH,-)4 33 (br mn 4114H, 2-H, 5-H), 5.30 (br. d, 11H, CHNH), 5.56 (br.s, 1H, 4-H) ppm

[0300]¹³C-NMR (CDC13, 75 MHz) δ=27.7.28.9, 28.0 [3q, C(CH,,)₃], 31.3 (t,3-CH₂), 52.0 (d. CHNH). 55.3, 56.9 (2t, C-2, C-S), 80 0, 80 9 [2s,(C(CH₃)₃], 12 1.1 (d, C-4), 133 6 (s, C-3), 153.2 (s, N═C—N), 155 2 (brs, NCOO), 176 5 (s, COOH) ppm

[0301] f) Ada-Gly-Asp-Tyr

[0302] The title peptide was prepared in same manner as example 8f)starting from 50 mg (0.03 mmol) Fmoc-L-Tyr-TCP resin. Instead ofTroc-Ada(Boc₂)-OH Boc-Ada(Boc2)-OH from example 12e) was used for theN-terminal amino acid. 12 mg of Ada-Gly-Asp-Tyr.HCl were obtained aswhite lyophilisate Amino acid analysis: Gly 1 05 (1), Asp 0.96 (1), Tyr1.00 (1 ), peptide content. 61 1%

[0303] pos. LSIMS: m/z 534.3 MH⁺

EXAMPLE 11 Ada-Gly-Asp-Phe-NH₂

[0304] The title compound was synthesized by solid-phase methodology ona ACT90 automated peptide synthesizer (Advanced ChemTec, Louisville,Ky.) using tritylchloride-polystyrene(1%)divinylbenzene (TCP; loading.0.96 mmol/g; PepChem, Tubingen) and Fmoc-Asp-Phe-NH₂ (NovaBiochem,Laufelfingen) as starting materials The Fmoc-protected dipeptide amide(320.4 mg, 0.72 mmol) was dissolved in 4 ml dichloromethane After i eqN-methyl-morpholin (NMM) was added the solution was given to 444 mg (048 mmol) dry TCP resin After 5 min an additional volume of 130 ul NMMwas added yielding a total amount of 1 80 mmol NMM in solution Themixture &as shaken for further 60 min Then the residual tritylchloridegroups were capped by the addition of 0 5 ml methanol After further 20min the resin was filtered off and washed with dichloromethane, DMF andmethanol. The resin was dried under reduced pressure. Loading of theresin was determined to be 0 057 mmol/g. The Fmoc group was removed bytreatment with piperidine/dimethylformamide (1 1 v/v) for 2×10 minAfterwards Fmoc-Gly-OH was coupled within 30 min in 30-fold excess in adouble coupling procedure using 1 eq TBTU and 1 eq NMM as activingagents. After removal of the Fmoc group by the same procedure asdescribed above Boc-Ada(Boc₂)-OH was coupled manually in DMF within 17 hby using a 25-fold excess of the protected amino acid and equimolaramounts of TBTU and NMM for activation. Cleavage from the resin anddeprotection of the peptide was performed according to example 8t)Instead of heptane trifluoroethanol (2 ml) was used to dissolve thedeprotected peptide From this solution the crude peptide wasprecipitated by the addition of 16 ml diethylether After centrifugationthe supernatant was discarded and the precipitate lyophilized from tert-butanol/water (1 1 v/v) Ada-Gly-Asp-Phe-NH₂.HCl (28 mg) was obtained aswhite lyophilisate.

[0305] Amino acid analysis: Gly 1.05 (I), Asp 1 03 (I); Phe 0.97 (I);peptide content 67 4%

[0306] pos. LSIMS: m/z 502 3 MH⁺

EXAMPLE 12 Description of the Pharmacological Experiments

[0307] Thrombin Time

[0308] A common test in clinical coagulation diagnostics is the thrombintime. This parameter detects the action of thrombin on fibrinogen andthe formation of blood clots. Inhibitors of thrombin result in anincreased thrombin time

[0309] In order to obtain plasma 9 parts fresh blood from healthy donorsare mixed with one part sodium citrate solution (0 11 mol/l) andcentrifuged at ca 3000 rpm for 10 min at room temperature, The plasmawas removed by pipette and can be stored for ca 8 h at room temperature.

[0310] 200 μl citrate plasma was incubated for 2 min at 37° C. in asphere coagulometer (KC10 from the Amelung-Company). 10 μldimethylsulfoxide (DMSO) or a solution of the active substance in DMSOwas added to 190 μl preheated thrombin reagent (Boehringer Mannheim,GmbH. contains ca. 3 U/ml horse thrombin and 0 0 12.5 M Ca2+) Astopwatch was started when this 200 μl solution was added to the plasmaand the time point at which coagulation starts was determined In thecontrol measurements the thrombin time was ca 24 seconds and wasincreased by the compound of example 3 depending on the concentration(test concentration/increase of thrombin time 5 μM>300*sec: 0 5 μM/96sec. 0 05 μM/9 sec) [* the experiment was stopped after 5 minutes]

[0311] Thrombin Inhibition

[0312] The kinetic measurements were carried out in 0 1 M phosphatebuffer which contained 0 2 M sodium chloride and 0 5 % polyethyleneglycol 6000 at a pH=7 5 and 25° C. using the substrateH-(D)-Phe-Pro-Arg-pNA (S-2238 Kabi) and human thrombin (Sigma specificactivity=2150 NIH-units/mg) in polystyrene semimicrocuvettes in a totalvolume of 1 ml

[0313] In a preexperiment is was determined whether the compound offormula (1) inhibits thrombin rapidly or slowly For this the reactionwas started first by adding 0 03 NIH units thrombin to a 100 μM solutionof the substrate and the active substance In a second experimentsubstrate was added to a solution of thrombin and the active substancewhich had been incubated for i min The increase of the concentration ofp-nitro-anilide with time was monitored spectrophotometrically for 12min at 405 nm (UV-VIS spectrophotometer Lambda-2 from the Perkin-ElmerCompany). Since the measurement curves obtained in both experiments werelinear and parallel, the active substance of formula (I) is a rapidthombin inhibitor. The inhibition constant Ki was determined as followsThe substrate was added at concentrations of 100 μM. 50 μM. 30 μM, 20 μMand at each substrate concentration a measurement was carried outwithout inhibitor and three measurements were carried out in thepresence of various concentrations of the inhibitor of formula (I). Thereactions were started by addition of thrombin. The increase inabsorbance at 405 nm caused by the formation of p-nitroanilide wasmonitored for a time period of 12 min. Measurement points (time versusabsorbance) were transferred to a PC at intervals of 20 sec. The ratesVo (changes in absorbance per sec. measurements without inhibitor) andVi (measurements with inhibitor) were determined front the data bylinear regression Only that part of each measurement was used in whichthe substrate concentration had been reduced by less than 15%. From onemeasurement series (constant inhibitor concentration, variable substrateconcentrations) Km′ and Vmax were determined by a non-linear fit to theequation$V = \frac{V\quad \max*\lbrack S\rbrack}{\lbrack S\rbrack + {K\quad m^{\prime}}}$

[0314] Finally Ki was determined from the total series of measurementsby non-linear fit to the equation$V = \frac{V\quad \max*\lbrack S\rbrack}{K\quad m*\left( {1 + {{\lbrack S\rbrack/K}\quad i} + \lbrack S\rbrack} \right.}$

[0315] The Michaelis constant Km was 3.8+2 μM in all measurements. Thecompound of example 4 inhibits thrombin.

[0316] Inhibition of Trypsin

[0317] 10 mg bovine pancreatic trypsin (Sigma) was dissolved in 100 mlmM hydrochloric acid and stored in a refrigerator 20 μl thereof wasadmixed with 980 ul 1 mM hydrochloric acid. 25 μl thereof was used foreach measurement. The measurement was carried out as described forthrombin. Km=45 μM. The compound of example 1 inhibits trypsin with aninhibition constant Ki of 100 nM.

[0318] GpIIb/IIIa Inhibition

[0319] The GpIlb/Illa fibrinogen Elisa is a modification of assays whichare described in the following literature: Nachman, R. L. & Leung, L. L.K. (1982): J. Clin. Invest. 69: 263-269 and Wright, P. S. et al. (1993):Biochem. J. 293:263-267.

[0320] Microtitre plates were coated overnight with 2 μg/ml isolatedactivated GpIIb/IIIa receptor. After unbound receptor had been removedby washing several times the surface of the plates is blocked with 1%casein and it is washed again. The test substance is added at therequired concentrations and the plates are incubated for 10 minuteswhile shaking. The natural ligand of the gpIIb/IIIa receptor,fibrinogen, is added. After incubating for 1 hour unbound fibrinogen isremoved by washing several times and the bound fibrinogen is determinedby means of a peroxidase-conjugated, anti-fibrinogen monoclonal antibodyby measuring the optical density at 405 nm in an ELISA instrument. Theinhibition of a fibrinogen-GpIIb/IIIa interaction results in a lowoptical density. The IC50 value was determined by a concentration/effectcurve. Compound gpIIb/IIIa-Inhibition IC₅₀ [μM] Example 7 14.4 Example 80.4 Example 9 2.3 Example 10 2.4 Example 11 12.8

1. Compounds of formula I

wherein R₁, R₂ can be the same or different and denote hydrogen, an α- ,β- , γ- or ω-amino acid or a derivative thereof, a peptidyl residue with1-50 amino acids, C₁-C₆-alkylsulfonyl, a C₆-C₁₄-arylsulfonyl residue, ora 5 or 6 membered heteroaryl with 1, 2 or 3 heteroatoms, Y denoteshydrogen or a residue of formula COX, X denotes hydrogen, an OR₃ orNR₁R₂ residue wherein R₃ denotes hydrogen or C₁-C₆-alkyl and R₁′, R₂′can be the same or different and have the meanings of the residues R₁and R₂ and their optically active isomers as well as pharmacologicallyacceptable salts or prodrugs thereof.
 2. Pharmacological compositionscontaining at least one compound of general formula I as claimed inclaim I in addition to conventional carrier and auxiliary agents.
 3. Useof compounds of formula I as claimed in claim 1 for the production of apharmaceutical composition for the treatment of diseases which are dueto thromboembolic events, osteoporosis, inflammations or tumourdiseases.
 4. Compound of formula I

wherein Y denotes H or COOH or its derivatives wherein the primary aminogroup, carboxylic acid group and amidino group are optionally protected.5. Use of a compound of formula I of claim 4 for the production of abiologically active substance comprising the backbone structure offormula I′ or I″


6. Use of the backbone structure of formula I′ or I″

to substitute an arginine or a known arginine mimetic structure in apharmaceutical active compound.
 7. Use of the backbone structure offormula I′ or I″

to substitute arginine in a peptide wherein the peptidyl residues at theamino or carboxyl group have, independently of each other, an amino acidchain length between 0 and 50 amino acids.